Research during the past two decades has suggested that the retinoblastoma tumor suppressor (pRB) and two related proteins (p107 and p130;collectively referred to as pocket proteins) play a fundamental role in regulating the cell cycle, and pRB has been shown to be a prototypical tumor suppressor mutated in a large portion of human tumors. In addition, pRB has been shown to play a pivotal role in differentiation of several tissues, including muscle and bone. Our work during the previous funding period, under the auspices of our parent grant application, has highlighted unique roles for each of the pocket proteins in responding to growth arrest cues and in both promoting and maintaining differentiation of muscle. In addition, we have succeeded for the first time in purifying pRB complexes from proliferating and differentiated muscle cells. In this grant, we propose the following aims to further dissect the mechanisms underlying pocket protein involvement in cell cycle exit and differentiation. (1) We will characterize pRB complexes in proliferating and differentiated cells and examine the impact of depleting associated proteins on gene expression and differentiation;(2) we will identify targets of the pRB complexes in cultured cells and in muscle tissue and examine the role of pRB complexes in modification of target gene chromatin;and (3) we will examine whether mechanisms analogous to those discovered in Aims 1 and 2 pertain to reversible growth arrest as well and determine if there are mechanisms that distinguish reversible and irreversible cell cycle exit. In the current Revision to the original parent application, we propose broadening our investigation of chromatin modifications associated with myogenic differentiation, focusing in particular on those changes that are dependent on the pRB family of proteins. We will incorporate the new, state-of-the-art ChIP-sequencing approach to examine these modifications in an unbiased, genome-wide manner. These studies will enhance our understanding of regulatory controls that are essential for both reversible and permanent withdrawal from the cell cycle and differentiation. In addition, they will elucidate critical interactions between repressors, co-repressors, and chromatin modifications as they occur in a developmentally relevant setting. PUBLIC HEALTH RELEVANCE: Cancer results in some cases when cells fail to properly differentiate. Differentiation is coupled to exit from the cell cycle, and the retinoblastoma (pRB) tumor suppressor plays a pivotal role in controlling growth arrest and differentiation. This proposal seeks to understand the underlying mechanisms whereby pRB regulates gene expression and controls the decision to permanently stop dividing and to terminally differentiate.